Allergies are caused by the immune reaction to commonly harmless proteins, allergens. Allergic diseases are reaching epidemic proportions all over the world. More than 25% of the population in industrialized countries suffer from type I allergy and the number is steadily increasing. Birch pollen allergy is a very common form of type I allergy. Bet v 1 is the major allergen of birch pollen. More information on the Bet v 1 allergen, its isoallergens and variants, is found on the WHO website www.allergen.org.
Type I allergy is based on the formation of immunoglobulin E (IgE) antibodies. The symptoms occur when an allergen molecule binds to two IgE antibodies bound to receptors on a mast cell or basophile surface and induces cross-linking of the IgE-FcεRI complexes. This triggers the degranulation of biological mediators, such as histamine and lipid mediators, which cause inflammatory reactions and symptoms, such as allergic asthma, rhinitis, food and skin allergy, and even anaphylaxis.
The IgE is a large molecule that consists of two identical light and heavy chains. There are five domains in the heavy chain of IgE: VH, Cε1, Cε2, Cε3 and Cε4. The size of the complete IgE molecule is about 200 kDa. The crystal structures of the Cε2-Cε4 fragment bound to its FcεRI receptor and the Cε2-Cε4 fragment have been determined (Garman et al., Nature 2000(406):259-266, and Wan et al., Nature Immunology, 2002(3):681-686).
In the last few years, the three-dimensional structures for a large number of different allergens have been determined. Structurally, these allergens vary considerably, and no common structural motif that could explain the capability of allergens to cause the production of IgE antibodies has been identified. However, there are studies implicating that allergenicity is restricted to only a few protein families, thus raising evidence that structural features of proteins could also have a role in allergenicity (Jenkins et al., J. Allergy Clin. Immunol. 2005(115):163-170; Raudauer et al., J Allergy Clin Immunol. 2008(121):847-852; Rouvinen et al., PloS ONE 2010(5):e9037).
The essential question when studying allergenicity involves the so-called B-cell epitope, the IgE antibody-binding site of an allergen. Unfortunately, however, this B-cell epitope cannot be deduced directly from the three-dimensional structure of an allergen. Additionally, there are differences in the epitopes of a defined allergen recognized by individual patient' IgE. Therefore, B-cell epitopes have been sought using various techniques and various basis, such as by analyzing allergenic fragments or peptides, which react with polyclonal IgE serum pools from allergic patients, site-directed mutagenesis of allergens, use of epitope mimics (mimotopes) and bioinformatics modeling studies. However, as yet, no general maps of dominating epitopes exist for any allergen.
With regard to birch pollen, for instance, Holm et al. (The Journal of Immunology 2004 (173): 5258-5267) produced Bet v 1 mutants containing 4 and 9 point mutants with the aim to manipulate surface topology in “selected areas”. The paper does not describe how this “selection” has been made. The mutants with four amino acid substitutions represented three different areas on the molecular surface and the mutants with nine amino acid substitutions represented five different areas on the molecular surface. These Bet v 1 mutants had in some cases reduced capacity to bind human serum IgE and to trigger human basophile histamine release. They were also able to induce IgG antibodies against unmutated Bet v 1.
Further modified recombinant allergens have been reported:
International patent publications WO 02/40676 and WO 03/096869 disclose numerous mutant forms of birch pollen allergen Bet v 1. These mutants were produced by introducing random mutations in the putative IgE binding site, based on sequence analysis of conserved surface structures of the Bet v 1 polypeptide. WO 03/096869 discloses the use of four primary mutations on different “small groups” on the allergen surface.
International patent publication WO 2007/073907 discloses a Bet v 1 polypeptide comprising three amino acid substitutions or deletions at amino acid sites 54, 115 or 123. There is no evidence that these mutants have reduced histamine release capacity.
International patent publication WO 2009/024208 discloses a Bet v 1 mutant having at least four mutations in the area amino acids 100-125. However, due to the mutations the tree-dimensional structure of the polypeptide is lost, and there is no reported histamine release activity.
International patent publication WO 2008/092992 discloses a method of blocking the type I surface interaction of allergenic substances by modifying amino acid residues on non-continuous allergenic epitopes, i.e., on a planar surface with an area of 600-900 Å on the allergenic substance and suggests that hypoallergenic birch pollen proteins could be prepared accordingly.
Niemi et al., Structure 2007(15): 1413-21, disclose one approach in the search of specific allergen epitopes in the line with the disclosure of Laver et al., Cell 1990(61):553-556, who state that the only rational method by which to determine the complete epitope of any allergen involves measuring crystal structure of an allergen in complex with an IgE antibody. Niemi et al. disclose the crystal structure of an IgE Fab fragment in complex with β-lactoglobulin (BLG). They also show how two IgE/Fab molecules bind the dimeric BLG and that the IgE epitope is different when compared to known IgG epitope structures, being a “flat” surface located in the β sheet region.
Rouvinen et al., PloS ONE 2010(5):e9037 investigated the role of dimeric structures of allergens using bioinformatics methods combined with native mass spectrometry. (Electrospray Ionization Fourier Transform Ion Cyclotron Mass Spectrometry, ESI FT-ICR-MS). The ESI-MS measurements of the 55 known crystal structures of allergens showed that 80% of them exist in symmetric dimers or oligomers in crystals and that the majority is transient dimers that are formed at high protein concentrations. The possible relationship between dimeric structure and allergenicity was studied with a recombinant allergen from cow's milk, β-lactoglobulin (rBos d 5 B), which occurs as a dimer, and its mutant H146P, which occurs mainly as a monomer. A somewhat reduced histamine releasing capacity was observed with the monomeric rBos d 5 B mutant H146P when compared to the native Bos d 5 B and recombinant Bos d 5 B. Although the authors conclude that dimerization could be a very common and essential feature for allergens and generally suggest that the preparation of purely monomeric variants of allergens could open up novel possibilities for specific immunotherapy, the ultimate role of structural features in allergenicity remain unknown. From the in vitro crystal analysis direct conclusions as to how the allergen molecules behave in a human body, i.e. in vivo, cannot be drawn.
Today the trend in the treatment of all allergic symptoms is towards an active induction of tolerance using allergen-specific desensitization instead of avoiding the allergen, which is often not possible, or merely treating the symptoms. Current desensitization therapy is based on allergens purified from natural sources, wherein batch to batch variations may lead to problems related to finding and maintaining the right dosage and efficiency of the treatment. These problems may lead to a potential risk of anaphylactic side effects and sensitization to new allergens.
The use of recombinant allergens for desensitizing would remove the disadvantages related to batch to batch variations, and the first recombinant allergens are in clinical trials (Valenta et al., Annu Rev Immunol 2010(28):211-41). The efficiency of such allergens in the clinic thus remains to be seen.
There is a recognized and large need for safe and efficient vaccines and therapy products to meet the increasing medical problem of allergy. At present the market for safe and efficient therapies of allergy is underdeveloped.